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NEW LC/MS/MS METHOD FOR THE QUANTIFICATION OF PHENYTOIN IN HUMAN PLASMA
Under the experimental conditions used, the lower limit of quantification
(LOQ) was of 2 µg/ml phenytoin. LOQ is the lowest amount of analyte
which can be measured with accuracy and precision less than 20%.
CONCLUSIONS
A simple, sensitive, accurate and precise HPLC/MS/MS method for
determination of phenytoin in human plasma using a simple single-step
extraction procedure is reported. Another advantage of the method is the
short chromatographic runtime of only 2.1 min. The method is suitable for
therapeutic drug monitoring studies and can also be used for pharmacokinetic
studies conducted on healthy volunteers [9-13].
EXPERIMENTAL SECTION
Reagents
Phenytoin, methanol, ammonium acetate, were purchased from Merck
(Merck KgaA, Darmstadt, Germany). Solvents used were HPLC grade and
all other chemicals were of analytical grade. Distilled, deionised water was
produced by a Direct Q-5 Millipore (Millipore SA, Molsheim, France) water
system. The drug-free human plasma was supplied by the Local Bleeding
Centre Cluj-Napoca, Romania.
Preparation of standard solutions
A stock solution containing 10 mg/ml phenytoin was prepared in
methanol. A working solution of 200 µg/ml was prepared by diluting the
appropriate volume of stock solution with plasma. Than this was used to
spike different volumes of drug-free plasma, providing finally eight plasma
standards with concentration between 2.0 and 80.0 µg/ml. Quality control
samples (QC) of 5.0, 32.0, and 64.0 µg/ml were prepared by diluting specific
volumes of working solution with plasma and were used to evaluate precision
and accuracy of the method.
Chromatographic and mass spectrometry systems and conditions
The HPLC system was an 1100 series model (Agilent Technologies)
consisted in a binary pump, an in-line degasser, an autosampler, a column
thermostat and an Ion Trap VL mass spectrometer detector (Bruckner Daltonics
GmbH, Germany). Chromatograms were processed using QuantAnalysis
Software. The detection of phenytoin was in MRM (MS/MS) mode, using an
electrospray positive ionization (ESI positive). The ion transitions monitored
was: m/z 253.1→(m/z 182.1 + m/z 225.1). Chromatographic separation was
performed at 45ºC on a Zorbax SB-C18 100 mm x 3 mm, 3.5 µm column
(Agilent Technologies), protected by an inline filter.
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